Recent observations of folate pool dynamics in cells treated with trimetrexate or methotrexate have revealed different folate depletion responses in breast and colon cancer cell lines than in hepatoma lines. An explanation of this difference is being sought in terms of the biochemical kinetics of the folate cycle. Differences seem unlikely to arise from dissimilar Michaelis and inhibition constants, since known values do not vary greatly across the cell lines. On the other hand, maximum enzyme activities may vary across cell type and are suspected of accounting for different folate dynamics. At present, elevated activities of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide transformylase (GT) are being examined for their ability to account for the different folate depletion rates observed in the various cell lines. Our folate cycle model (JBC 264 10552 1989) has been employed to show that ten-fold TS activity increases will deplete reduced folates down to experimental assay limits. The model has also shown that similar increases of DHFR activity will tend to offset any depletion caused by increased TS activity. With model calculations as a guide, colon and breast cell lines with order-of-magnitude TS increases are being investigated experimentally in the MB/NCI to verify the predicted depletion. In addition, DHFR, GT, and formyl-dihydrofolate synthetase activities are being monitored for interference effects suggested by the folate cycle model.